1. Field of the Invention
Embodiments of the invention relate to compositions and methods for transepithelial delivery of peptides with incretin hormone activity for the treatment of chronic disease conditions, particularly diabetes mellitus. Oligopeptides are used to facilitate topical delivery.
2. Description of the Related Art
Diabetes mellitus is a group of metabolic diseases characterized by elevated blood sugar levels (hyperglycermia). Hyperglycermia is a result of absolute or relative insufficiency of insulin secretion or resistance to insulin action or both. The majority of diabetes cases fall into two categories: type 1 diabetes and type 2 diabetes. Type 2 diabetes is the most common form of diabetes, accounting for 90% of cases. An estimated 16 million Americans have type 2 diabetes. Type 2 diabetes is usually caused by resistance to insulin action in the setting of inadequate compensatory insulin secretory response. (c.f. Jay S. Skyler “Diabetes Mellitus: Pathogenesis and Treatment Strategies” Journal of Medicinal Chemistry, 2004, vol. 47, 4113-4117.)
Insulin is a key player in the control of carbohydrate and lipid metabolism. When glucose is liberated from dietary carbohydrate and absorbed into the blood, elevated concentrations of blood glucose stimulate release of insulin. Insulin facilitates entry of glucose into muscle, adipose and several other tissues. Insulin also stimulates the liver to store glucose in the form of glycogen. As blood glucose concentrations fall, insulin secretion ceases. In the absence of insulin, the cells in the body will switch to using alternative fuels like fatty acids for energy, and subsequently, enzymes will break down the glycogen in the liver.
Insulin also has important effects on lipid metabolism. Insulin promotes synthesis of fatty acids in the liver. When the liver is saturated with glycogen, any additional glucose taken up by hepatocytes is shunted into pathways leading to synthesis of fatty acids, which are exported from the liver as lipoproteins. Insulin also inhibits breakdown of fat in adipose tissue. From a whole body perspective, insulin has a fat-sparing effect. Not only does it drive most cells to preferentially oxidize carbohydrates instead of fatty acids for energy, insulin indirectly stimulates accumulation of fat in adipose tissue.
Because of the important role insulin plays in the control of carbohydrate and lipid metabolism, derangements in insulin secretion and action have widespread and devastating effects on many organs and tissues. Diabetes mellitus, the most important metabolic disease of man, is an insulin deficiency state. Type 1 or insulin-dependent diabetes mellitus is the result of an immune-mediated destruction of pancreatic islet β-cells with consequent insulin deficiency and the need to replace insulin. Type 2 or non-insulin-dependent diabetes mellitus is a syndrome of insulin resistance, in which target tissues fail to respond appropriately to insulin. Diabetes patients suffer the pervasive metabolic derangements that include altered metabolism of carbohydrates, fats and proteins. Over time, metabolic disruption may lead to long-term damage, dysfunction and failure of various organs, especially the eyes, kidneys, nerves, heart and blood vessels.
Current pharmacologic agents used to treat type 2 diabetes include insulin, biguanides, sulfonylureas and thiazolidinediones. Because of the natural progression of type 2 diabetes, most diabetes patients eventually require insulin therapy. The major drawbacks of these drugs include low blood glucose (hypoglycemia), weight gain and edema. In addition, none of these compounds offer the potential to preserve the function of insulin-producing β-cells in the pancreas.
Incretin hormones are hormones that cause an increase in the amount of insulin released when glucose levels are normal or, more particularly, when they are elevated. These incretin hormones have other actions beyond the initial incretin action defined by insulin secretion. They also have actions to reduce glucagon production and delay gastric emptying. They may also have actions to improve insulin sensitivity, and they may increase islet cell neogenesis, namely the formation of new islets. Incretin hormones augment insulin response when glucose is absorbed through the gut. There are two known incretin hormones in humans: glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). The therapeutic use of incretin hormones in treating conditions including diabetes, obesity, cardiovascular diseases and Alzheimer's disease is currently an area of intense research activity.
Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal peptide of 42 amino acids. GIP is released from duodenal endocrine K cells after absorption of glucose or fat. Glucagon-like peptide-1 (GLP-1) is synthesized in the L-cells of the lower intestinal tract in response to the presence of nutrients in the distal small intestine. Both GIP and GLP-1 potentiate glucose-induced insulin secretion from the pancreatic β-cells.
The important effects of the GIP and GLP-1 on carbohydrate and lipid metabolism have been demonstrated through experiments involving deleting the genes of their receptors. Knockout of the GIP receptor is associated with significant glucose intolerance. It has also been found that mice lacking GIP receptors are protected from both obesity and insulin resistance when being fed with a high-fat diet. Mice without GLP-1 receptors are glucose intolerant and exhibit fasting hyperglycemia.
GLP-1 is one of the most potent insulinotropic substances with half-maximal effective concentration on β-cells at 10 pmole/l. The insulinotropic effect of GLP-1 is strictly glucose dependent. GLP-1 stimulates all steps of insulin biosynthesis as well as insulin gene transcription, thereby providing continued and augmented supplies of insulin for secretion. GLP-1 has trophic effects on β-cells. It stimulates β-cell proliferation and enhances the differentiation of new β-cells from progenitor cells in the pancreatic duct epithelium. GLP-1 also inhibits both cytokine and fatty acid-induced apoptosis in β-cells. Patients with type 2 diabetes mellitus have significantly impaired GLP-1 secretion and impaired responsiveness of β cells to GIP. However, near normal insulin responses are restored in type 2 diabetes patients after GLP-1 injection. Therefore, GLP-1 represents an attractive antidiabetic agent.
Exendin peptides were isolated from the salivary glands of the lizard Heloderma species. They were so named for the reason that they were isolated from an exocrine gland and were subsequently shown to have endocrine actions. Exendin-3 was isolated from Heloderma horridum and exendin-4 was isolated from Heloderma suspectum. These two peptides share an identical sequence except for substitutions in residues 2 and 3 from the amino terminus, and both peptides can stimulate cAMP activity in dispersed pancreatic acinar cells. Exendin-4 is a potent agonist for the mammalian GLP-1 receptor. Exendin-4 is much more potent than native GLP-1 for the treatment of diabetes, largely due to its resistance to the aminopeptidase dipeptidyl peptidase IV (DPP-IV) mediated inactivation. In contrast to GLP-1 which contains an alanine at position 2, exendin-4 has a position 2 glycine; hence it is not a substrate for DPP-IV and has a much longer t1/2 in vivo.
In a study in which patients received infusion of natural GLP-1 via MiniMed insulin pumps for 6 weeks, it was demonstrated that GLP-1 can rapidly lower fasting blood glucose by 4-5 mM, mainly within the first week of treatment. (Zander, M.; Madsbad, S.; Madsen, J. L.; Holst, J. J. “Effect of 6-Week Course of Glucagon-Like Peptide 1 on Glycaemic Control, Insulin Sensitivity, and Beta-Cell Function in Type 2 Diabetes: A Parallel-Group Study”. Lancet 2002, 359, 824-830.) The potent glucose-lowering properties of GLP-1 and its analogs, including exendin-4 (exenatide, AC2993) from Lilly and Amylin (Stoffers, D. A.; Desai, B. M.; DeLeon, D. D.; Simmons, R. A. “Neonatal Exendin-4 Prevents the Development of Diabetes in the Intrauterine Growth Retarded Rat.” Diabetes 2003, 52, 734-740.), liraglutide (γ-L-glutamoyl(N-R-hexadecanoyl))-Lys26,Arg34-GLP-1(7-37), NN2211) from Novo Nordisk (Elbrond, B.; Jakobsen, G.; Larsen, S.; Agerso, H.; Jensen, L. B.; Rolan, P.; Sturis, J.; Hatorp, V.; Zdravkovic, M. “Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of a Single-Dose of NN2211, a Long-Acting Glucagon-Like Peptide 1 Derivative, in Healthy Male Subjects”. Diabetes Care 2002, 25, 1398-1404.), CJC-1131 (D-Ala8Lys37[2-[2-[2-maleimidopropionamido(ethoxy)ethoxy]acetamide-GLP-1(7-37)) from Conjuchem (Kim, J. G.; Baggio, L. L.; Bridon, D. P.; Castaigne, J. P.; Robitaille, M. F.; Jette, L.; Benquet, C.; Drucker, D. J. “Development and Characterization of a Glucagon-Like Peptide 1-Albumin Conjugates The Ability To Activate the Glucagon-Like Peptide 1 Receptor in Vivo”. Diabetes 2003, 52, 751-759.), ZP-10A from Zealand and Aventis (Petersen, J. S.; Thorkildsen, C.; Lundgren, K.; Neve, S. “ZP10: A New GLP-1 Agonist That Prevents Diabetes Progression and Increases Insulin mRNA Expression in Db/Db Mice”. Diabetologia 2002, 45, A147.) and BIM-51077 from Ipsen (Dong, J. Z.; Shen, Y.; Zhang, J.; Taylor, J. E.; Woon, C.; Morgan, B.; Skinner, S.; Cawthorne, M.; Culler, M.; Moreau, J. “Design and Synthesis of a Novel GLP-1 Analog, BIM-51077, Which Has Significantly Improved in Vivo Activity”. Biopolymers 2003, 71, 391.), have prompted consideration of its use for the treatment of patients with Type 2 diabetes. The above is summarized in Table 1 below.
TABLE 1Proprietory GLP-1 receptor agonistsGLPagonistChemical nameDeveloperReferenceExenatide,Exendin-4, seeLilly &Stoffers, et al.AC2993SEQ ID NO: 6Amylin((2003) Diabetes52: 734Liraglutideγ-L-glutamoylNovoElrond, et al.(N-R-hexadecanoyl)-Nordisk(2002) DiabetesLys26, Arg34-GLP-Care 25: 13981(7-37), NN2211CJC-1131D-Ala, 8Lys37[2-ConjuchemKim, et al. (2003)[2-[2-maleimido-Diabetes 52: 751)propionamido(ethoxy)-ethoxy]acetamide-GLP-1(7-37)ZP10ASee SEQ ID NO: 35Zealand &Petersen, et al.Aventis(2002)Diabetologia2002,45: A147BIM-51077IpsenDong, et al.(2003)Biopolymers71, 391
Drug delivery is the biggest challenge for the development of GLP-1 and its analogs into therapeutics. The compounds mentioned above are all peptides and have to be injected. Transit nausea and vomiting are the major side effects of this class of compounds and are associated with peak concentrations introduced by injection. These side effects limited the dosage of GLP-1 in human studies (Drucker, Daniel J. “Enhancing Incretin Action for the Treatment of Type 2 Diabetes” (October 2003) Diabetes Care vol. 26 (10): 2929-2940). Likewise, Nielsen, et al. report that the optimal glucose-lowering dose range for exenatide (synthetic exendin-4) is 0.05 to 0.2 μg/kg, but that nausea and vomiting are dose-limiting (Nielson, et al. (April 2003) “Pharmacology of exenatide (synthetic exendin-4) for the treatment of type 2 diabetes” Current Opinion in Investigational Drugs vol. 4 (4): 401-405). A need exists to provide a therapeutic dose of a diabetic drug treatment such as GLP-1 or an analog thereof, while minimizing unwanted side effects.
Drug delivery through the transdermal pathway offers clear advantages. First, transdermal delivery potentially improves patient compliance compared with oral delivery. Smaller doses may be used for the same drug, helping to minimize side effects such as nausea. Second, the problem of first-pass metabolism is avoided, as well as the peaks and valleys created by oral delivery and GI tract absorption. There are no restrictions around the time that the drug should be administered or whether the patient may eat afterward. In particular, multi-day patch delivery offers ease of use and is convenient, without the requirement to remember to take a medicine at a specific time. A final benefit of transdermal delivery versus oral or injection is that dosage may be stopped abruptly by simply removing the patch if adverse side effects are experienced.
As the largest organ in the human body, one of the most important functions of the human skin is to provide a physicochemical barrier to defend the body from the ingression of toxic chemicals and microorganisms. At the most coarse level, human skin is made of three layers. Stratum corneum, located on the outer surface of the skin, is a non-living layer of keratin-filled cells surrounded by a lipid-rich extracellular matrix that provides the primary barrier to drug delivery into skin. The epidermis below is a viable tissue devoid of blood vessels. Just below the dermal-epidermal junction, the dermis contains capillary loops that can take up transdermally administered drugs for systemic distribution. For most molecules the stratum corneum is the rate-limiting barrier to drug delivery. There are essentially three pathways by which a molecule can traverse intact stratum corneum. In the transappendageal route, a drug molecule takes advantage of the pores of hair follicles or sweat ducts to bypass the barrier of stratum corneum. In the transcellular route, a drug molecule penetrates across the stratum corneum through a series of events that include partition between bilayer lipid and keratin-filled cells followed by diffusion through the hydrated keratin. The intercellular lipid route provides the principal pathway by which most small, uncharged molecules traverse stratum corneum by moving through the continuous lipid domains between the keratinocytes.
Despite the success of transdermal delivery technology, the number of drugs that can be administered through the transdermal route is very limited. All of the drugs presently administered across skin share three constraining characteristics: low molecular mass (<500 Da), high lipophilicity (oil soluble) and small required dose (up to milligrams). There are a number of emerging technologies on the horizon that aim at removing these limitations for transdermal drug delivery. One of these technologies is the use of peptide oligomers with cell-penetrating properties as transdermal carriers. Cell-penetrating peptides are short polycationic oligomers that can penetrate across cell membranes through a receptor independent, non-endosome mechanism, potentially through interacting and disrupting of the bilayer lipid of the cell membrane. The same mechanism also potentially accounts for the transdermal activities of these peptides. In a recent paper, Rothbard et al. (Rothbard, J. B.; Garlington, S.; Lin, Q.; Kirschberg, T.; Kreider, E.; McGrane, P. L.; Wender, P. A.; Khavari, P. A. “Conjugation of arginine oligomers to cyclosporine A facilitates topical delivery and inhibition of inflammation”, Nature Medicine, 2000, 6, 1253-1257) conjugated a heptamer of arginine to cyclosporine A through a pH-sensitive linker to produce R7-CsA. In contrast to unmodified cyclosporine A, which failed to penetrate skin, topically applied R7-CsA was efficiently transported into cells of mouse and human skin. R7-CsA reached dermal T lymphocytes and inhibited cutaneous inflammation.
The present inventors have discovered transdermal delivery methods for administration of peptide drugs with incretin hormone activity, particularly GLP-1 or exendin-4. It has been demonstrated that near normal insulin responses could be restored in type 2 diabetes patients after GLP-1 injection. Therefore, GLP-1 and its more stable analog exendin-4 are attractive antidiabetic agents. These peptide drugs are useful in treating diabetes, obesity, cardiovascular diseases and Alzheimer's disease.
For the treatment of such chronic disorders, non-invasive and patient friendly drug delivery methods such as oral, nasal or transdermal are clearly more practical than injection or drug pump implantation. Of these, transdermal delivery has many advantages over oral or nasal delivery for the administration of GLP-1 or exendin-4. Orally delivered peptide/protein drugs are subjected to harsh conditions prior to absorption through the gastrointestinal tract. During absorption through the nasal mucosa considerable metabolism may occur. The present invention successfully conjugated exendin-4 with a known transdermal carrier arginine oligomer. Our biological data showed significant enhancement for the transdermal delivery of such conjugates as compared with exendin-4 alone.